Freeze-dried composite materials

ABSTRACT

The invention provides a sterile freeze-dried sponge, wherein at least 80% by weight of the sponge consists of collagen and oxidized regenerated cellulose in the weight ratio 60:40 to 40:60, wherein the sponge has a dry tensile strength of more than 3N and/or a wet tensile strength of more than 1 N. Preferably, the collagen has a degree of denaturation less than 20% and the sponge is substantially free of chemical cross-links. The invention also provides methods for the manufacture of sponges according to the invention

FIELD

[0001] The present invention relates to freeze-dried pads comprising amajor fraction of a mixture of collagen and oxidized regeneratedcellulose (ORC), and to processes for the production of such pads.

BACKGROUND

[0002] WO98/00180 describes the use of freeze-dried sponges of collagenadmixed with oxidized regenerated cellulose (ORC) for the treatment ofchronic wounds. Such sponges must in practice meet stringentrequirements of purity, sterility and non-antigenicity.

[0003] It has not hitherto been possible to provide sponges ofcollagen/ORC mixtures having high reproducibility and high tensilestrength both when wet and when dry. In particular, collagen is prone todenaturation when it is sterilized by gamma-irradiation. Furthermore,collagen sponges tend to disintegrate rather rapidly in wound fluid,especially in the presence of collagenase enzymes. Whilst this problemcan be diminished by chemical cross-linking of the collagen sponge usingcross-linking agents such as glutaldehyde, the use of such cross-linkingagents can give rise to problems of toxicity and antigenicity.

SUMMARY OF THE INVENTION

[0004] Accordingly, it is an object of the present invention to providephysiologically acceptable, sterile sponge pads based on collagen/ORCmixtures that exhibit high tensile strength.

[0005] It is a further object of the present invention to providephysiologically acceptable, sterile sponge pads based on collagen/ORCmixtures that have very high purity, sterility and low bioburden.

[0006] It is a further object of the present invention to providephysiologically acceptable, sterile sponge pads based on collagen/ORCmixtures that have high uniformity.

[0007] It is a further object of the present invention to providephysiologically acceptable, sterile sponge pads based on collagen/ORCmixtures that exhibit reduced resorption rates under simulatedphysiological conditions.

[0008] It is a further object of the present invention to providephysiologically acceptable, sterile sponge pads based on collagen/ORCmixtures that exhibit high mechanical strength and long resorption timeswithout chemical cross-linking.

[0009] The present invention provides a sterile freeze-dried sponge,wherein at least 80% by weight of the sponge consists of a mixture ofcollagen and oxidized regenerated cellulose in the weight ratio 60:40 to40:60, and wherein the sponge has a dry tensile strength as hereindefined of more than 3N.

[0010] The freeze-dried sponge pad is sterile. Preferably, the sterilityassurance level is better than 10⁻⁶. Preferably, the sponge has beensterilized by gamma-irradiation. The sponge comprises at least 80% byweight of a mixture of collagen and ORC in the rate ratio 60:40 to40:60. Preferably, the weight ratio contains a small excess of collagen,in a range 50:50 to 40:60 ORC:collagen. Preferably, the freeze-driedsponge consists essentially of collagen, ORC, water and up to 5% of oneor more therapeutically active substances such as growth factors.Preferably, the freeze-dried sponge contains no more than 1% by weightof constituents other than collagen, ORC and water.

[0011] The collagen content is determined by hydrolysing the collageninto its constituent amino acids and analyzing for hydroxyproline asdetailed below. The collagen content is calculated to be 7.19 times thehydroxyproline content. The ORC content is determined by hydrolyzing itto its constituent monosaccharides and analyzing for glucuronic acid asdetailed further below.

[0012] Preferably, the freeze-dried sponge has a pH, measured ashereinafter described, of from 2.3 to 4.0, preferably from 2.5 to 3.0.

[0013] Preferably, the sterile freeze-dried sponges according to thepresent invention have a degree of collagen denaturation, measured ashereinafter described, of less than 15%, preferably less than 10%, andmore preferably less than 5%. It is a particularly advantageous featureof the freeze-dried sponges according to the present invention that thecollagen is stabilized against denaturation by the gamma-irradiationused in the sterilizing. The degree of denaturation of the collagen isdetermined by treatment with trypsin to dissolve the denatured collagen(trypsin does not dissolve native collagen), followed by filtration andquantitation of the hydroxyproline in the filtrate, as detailed furtherbelow.

[0014] The sterile freeze-dried sponges according to the presentinvention preferably have a dry tensile strength (maximum load measuredas hereinafter described) greater than 3N, preferably greater than 4N.Preferably, the dry tensile load at 20% extension, measured ashereinafter described, is greater than 2.5N, preferably greater than3.5N. Preferably, the dry extension at break, measured as hereinafterdescribed, is from 15 to 30%, preferably from 20 to 25%.

[0015] The tensile strength characteristics of the sponges according tothe present invention are further characterised by wet strengthmeasurements on samples that have been soaked for 15 minutes in PBSprior to testing. The resulting wet strength maximum load is preferablygreater than 1N, more preferably greater than 1.25N. The wet load at 20%extension is greater than 0.1N, preferably greater than 0.2N, mostpreferably 0.2-0.3N. The wet extension at break is preferably 75-100%,more preferably 80-90%.

[0016] Preferably, the sterile freeze-dried sponges according to thepresent invention are not chemically cross-linked. They have may somedehydrothermal cross-linking as a result of the freeze-drying process,but preferably there is no chemical cross-linking by glutaldehyde or thelike. This reduces the antigenicity and processing costs of the sponges.The present invention achieves satisfactory physical properties of thesponges and sufficiently long resorption times in vivo by very carefulcontrol of the composition and manufacturing conditions of the sponges.In particular, the sponges preferably contain ORC fibers, wherein avolume fraction of at least 80% of the fibers have lengths in the rangeof 20 μm to 1000 μm. Such a size distribution can be achieved, forexample, by milling an ORC cloth, followed sieving the milled powder toremove fibers outside the range. Preferably, the average (mean byvolume) length of the ORC fibers is in the range 250 μm to 450 μm.

[0017] The selection of ORC fiber lengths in this range results in easymixing of the ORC and collagen and highly homogeneous products. The ORCis more thoroughly complexed with the collagen, which results inenhanced therapeutic properties of the sponge. Furthermore, the ORC ismore effective to reduce denaturation of the collagen by gamma-radiationduring sterilization. Surprisingly, these advantages can be achievedwhile maintaining the tensile strength of the sponge despite the smallsize of the ORC fibers.

[0018] The desired physicochemical properties of the freeze-driedsponges according to the present invention are further achieved by theuse of collagen that has undergone sequential alkali and acid treatmentsteps to purify the collagen substantially without denaturing thecollagen fibers. Preferably, the bioburden (TVC) of the freeze-driedsponge according to the present invention is less than 100 cfu/g, morepreferably less than 10 cfu/g, and most preferably less than 1 cfu/g.

[0019] The sterile freeze-dried sponges according to the presentinvention have high and uniform porosity, and a high liquid absorptioncapacity. The measured absorption of the uncompressed pads in 0.9%saline is preferably greater than 12 g/100 cm², more preferably greaterthan 15 g/100 cm^(2.)

[0020] Preferably, the sterile freeze-dried sponge according to thepresent invention has a resorption time under simulated physiologicalconditions as described in more detail below of more than 48 hours.

[0021] The present invention further provides a method of manufacture ofa freeze-dried sponge pad comprising the steps of:—

[0022] providing an acidified paste of purified collagen fibers, whereinthe collagen is less than 10% denatured;

[0023] providing oxidized regenerated cellulose fibers, wherein at least80% of said fibers have lengths in the range of 20 μm to 1000 μm;

[0024] combining said collagen and said ORC fibers in a homogeneousaqueous dispersion in a weight ratio of 60:40 to 40:60 collagen:ORC,said aqueous dispersion being acidified to a pH in the range of 2.8 to3.2 and having a total solids concentration of 0.8 to 1.2% by weight;

[0025] pouring said aqueous dispersion into trays to a depth greaterthan 1 cm;

[0026] freezing the dispersion to a temperature less than −30° C.,followed by a temperature programmed freeze drying and dehydrothermalcross-linking to a final moisture content of 5-15% by weight;

[0027] splitting the freeze-dried dispersion to remove surface layersand leave one or more pads; and

[0028] sterilizing the one or more pads by gamma-irradiation.

[0029] Preferably, the process according to the present invention iscarried out substantially without the use of any chemical cross-linkingagents.

[0030] Preferably, the step of providing collagen comprises thefollowing steps:—

[0031] providing fresh and unswollen splits of bovine corium;

[0032] treating the corium splits with a solution containing sodiumhydroxide and hydrogen peroxide to swell and sterilize the corium; then

[0033] treating the corium with a aqueous alkali solution at a pHgreater than 12 and temperature less than 50° C. for a period of 10-14days; then

[0034] treating the corium with a aqueous acid solution at a pH of0.8-1.2 and temperature less than 50° C. until the pH of the coriumsplits drops to less than 2.5; then

[0035] washing the corium, and comminuting the corium with sufficientwater to form a paste.

[0036] This treatment results in a collagen of exceptional purity anduniformity, without significant denaturing of the collagen. The collagenpaste may be stored in the frozen state, but preferably the collagen isnot freeze-dried intermediate the above steps and the step of combiningthe collagen with the ORC.

[0037] Preferably, the step of providing oxidized regenerated cellulosefibers comprising milling an oxidized regenerated cellulose cloth andscreening the milled particles to remove particles having size less than20 μm or greater than 1000 μm.

[0038] Preferably, the step of dispersing the collagen and the ORCcomprises the steps of:—

[0039] adding an acid-swollen collagen/water paste to acidified water;

[0040] adding oxidized regenerated cellulose fibers to the acidifiedwater; and

[0041] homogenizing the resulting mixture.

DETAILED DESCRIPTION

[0042] The dispersion is poured into trays to a depth of at least 10 mm,preferably at least 20 mm, and frozen into blocks in the trays beforefreeze drying. The freezing is preferably carried out by placing thetrays containing the slurry onto pre-chilled shelves at −55° C. Thetrays are then loaded into a freeze-dryer, held at −50° C. for twohours, then at −40° C. before starting the freeze-drying cycle. Thisfreezing method gives more uniformly distributed ice crystals, and hencemore uniform products, than simply blast freezing the slurry in thetrays.

[0043] Preferably, the step of freeze drying is carried out withdehydrothermal cross-linking using a temperature program in the range−40° C. to ×30° C., to give blocks of freeze dried material. The blocksare split to remove surface layers, and to provide one or more pads. Thesettling of collagen and ORC fibers in trays results in a desiredorientation of collagen and ORC fibers in the final pads. Furthermore,splitting the final pads from a larger block ensures that they have highhomogeneity and surface uniformity.

[0044] Preferably, the step of sterilizing is carried out bygamma-irradiation at a dose of 18-29 KGy. It has been found thatsurprisingly little denaturation of the collagen takes place in thesterilizing step, which may be due to a stabilizing effect of the ORC.

[0045] In preferred embodiments of the process according to the presentinvention, the weight ratio of collagen to oxidized regeneratedcellulose is from 50:50 to 55:45 and the pH of the aqueous dispersion isfrom 2.9 to 3.1.

[0046] A specific embodiment of the process and product according to thepresent invention will now be described further, by way of example.

EXAMPLE 1

[0047] A freeze-dried collagen/ORC sponge is prepared as follows.

[0048] First, the collagen component is prepared from bovine corium asfollows. Bovine corium is split from cow hide, scraped and soaked insodium hypochlorite solution (0.03% w/v) to inhibit microbial activitypending further processing.

[0049] The corium is then washed with water and treated with a solutioncontaining sodium hydroxide (0.2% w/v) and hydrogen peroxide (0.02% w/v)to swell and sterilize the corium at ambient temperature.

[0050] The corium splits then undergo an alkali treatment step in asolution containing sodium hydroxide, calcium hydroxide and sodiumbicarbonate (0.4% w/v, 0.6% w/v and 0.05% w.v, respectively) at pHgreater than 12.2, ambient temperature, and for a time of 10-14 days,with tumbling, until an amide nitrogen level less than 0.24 mmol/g isreached.

[0051] The corium splits then undergo an acid treatment step with 1%hydrochloric acid at ambient temperature and pH 0.8-1.2. The treatmentis continued with tumbling until the corium splits have absorbedsufficient acid to reach a pH less than 2.5. The splits are then washedwith water until the pH value of corium splits reaches 3.0-3.4.

[0052] The corium splits are then comminuted with ice in a bowl chopperfirst with a coarse comminution and then with a fine comminutionsetting. The resulting paste, which is made up in a ratio of 650 g ofthe corium splits to 10 g of water, as ice, is frozen and stored beforeuse in the next stage of the process. However, the collagen is notfreeze-dried before admixture with the ORC in the next stage.

[0053] The ORC component of the freeze-dried pad is prepared as follows.A SURGICEL cloth (Johnson & Johnson Medical, Arlington) is milled usinga rotary knife cutter through a screen-plate, maintaining thetemperature below 60° C.

[0054] The milled ORC powder and the required weight (according tosolids content) of frozen collagen paste are then added to a sufficientamount of water acidified with acetic acid to obtain a pH value of 3.0and a total solids content of 1.0%. The mixture is homogenized through aFryma MZ130D homogenizer, progressively diminishing the settings to forma homogeneous slurry. The pH of the slurry is maintained at 2.9-3.1. Theslurry temperature is maintained below 20° C., and the solids content ismaintained at 1% ±0.07.

[0055] The resulting slurry is pumped to a degassing vessel. Vacuum isinitiated for a minimum of 30 minutes, with intermittent stirring, todegas the slurry. The slurry is then pumped into freeze-drier trays to adepth of 25 mm. The trays are placed onto freezer shelves where thetemperature has been preset to −40° C. The freeze-drier programme isthen initiated to dry and dehydrothermally cross-link the collagen andORC to form thick sponge pads.

[0056] On completion of the cycle, the vacuum is released, thefreeze-dried blocks are removed, and are then split to remove the topand bottom surface layers, and to divide the remainder of the blocksinto 3 mm-thick pads. The step of splitting the freeze-dried blocks intopads is carried out with a Fecken Kirfel K1 slitter.

[0057] Finally, the pads are die-cut to the desired size and shape on adie-cutter, packaged, and sterilized with 18-29 KGy of cobalt 60gamma-irradiation. Surprisingly, this irradiation does not causesignificant denaturation of the collagen, which appears to be stabilizedby the presence of ORC.

[0058] The resulting freeze-dried collagen ORC pads have a uniform,white, velvety appearance. The thickness of the pads is 3.2±0.17 mm (N=8batches). The collagen content is 54%±3.8% (N=12 batches). Thehydroxyproline content is 7.6±0.5% (N=12 batches). The carboxylatecontent is 10.98±0.81% (N=12 batches). The ash content is 0.16±0.1%(N=12 batches). The heavy metals (lead) content is less than 1 ppm. ThepH is 2.78±0.15. The denaturation level is 4.87±1.54%. The endotoxinlevel is 33.5±0.9 cfu/g. The bioburden level is 0.2±0.3 cfu/g. Themoisture content (loss on drying) is 12.0±12.8%.

[0059] Procedure 1

[0060] The collagen content of the materials according to the presentinvention is measured as follows:—

[0061] Briefly, collagen is hydrolysed into constituent amino acids. Theamount of the amino acid hydroxyproline is determined by oxidizing withchloramine-T and then coupling with 4-dimethylamino-benzaldehyde toproduce a coloured product, the concentration of which is measuredspectophotometrically at 550 nanometers.

[0062] Hydrolysis of the samples is carried out with 6 molarhydrochloric acid at 105° C. until digestion is completed, which takesat least 16 hours. The solution is then neutralized to pH 6 with 6 molarNaOH solution. The solution is then diluted. Typically, for a 10 mgsample, the procedure uses 1 ml of 6 molar HCl, and the final volume foranalysis on dilution is 500 ml.

[0063] A 1.0 ml sample of the test solution is treated with 1.0 ml of anoxidant solution prepared by dissolving 7 gm of chloramine-T in 600 mlof citrate buffer. The mixture is allowed to stand for 10 minutes, afterwhich 1.0 ml of 20% perchloric acid is added, mixed and allowed to standfor 5 minutes at room temperature.

[0064] The mixture is then treated with 1.0 ml of a colour reagentprepared by dissolving 30 gm of 4-dimethylamino benzaldehyde in 45 ml ofperchloric acid (60% w/v) followed by dilution in 250 ml ofpropane-2-ol. The mixture was treated in a water bath at 60° C. for 20minutes, cooled for 5 minutes, followed by reading the optical densityat 550 nanometers. The optical density is compared against valuesmeasured for control samples of pure collagen at various concentrations,pure hydroxyproline at various concentrations, and blank control samplesto arrive at the hydroxyproline content.

[0065] The collagen content of the sample in weight percent is obtainedby multiplying the measured hydroxyproline content in weight percent by7.19.

[0066] Procedure 2

[0067] The amount of denatured collagen present in the materialsaccording to the present invention is determined as follows.

[0068] Briefly, native collagen is protected by its triple-helicalstructure against proteolytic enzymes except for specific collagenases.If the helical structure is damaged, the resulting denatured collagen issusceptible to other proteases, such as trypsin, and is degraded topeptides. In this procedure, trypsin-resistant native collagen isseparated from the degraded peptides by salt precipitation, andnon-native collagen present in the filtrate is quantitated byhydroxyproline analysis.

[0069] A sample of the material according to the invention (100 mg) isweighed into a 50 ml conical flask. To the flask is added 10 ml oftris-HCl buffer solution containing 500 units trypsin. Blank experimentswithout the trypsin enzyme are also run. The mixtures are shaken at 4°C. for 5 hours. Then 2.5 ml of 25% NACl in 3 molar acetic acid are addedto each container and mixed thoroughly. The containers are then placedin a refrigerator at 4° C. for a minimum of 16 hours. The chilledextract is filtered through Whatman 541 filter paper into 50 ml beaker,and the hydroxyproline content of the sample of the filtrate is measuredby the method according to Procedure 1. Denatured collagen is calculatedas 7.19× the measured hydroxyproline level, and the percentage ofdenatured collagen is calculated by comparison with the total collagencontent measured by Procedure 1.

[0070] Procedure 3

[0071] The ORC content of materials according to the present inventionis measured by a method similar to that described by Bitter and Muir inAnalytical Chemistry vol. 4 (1962), pages 300-334.

[0072] Briefly, the material is hydrolysed to its individualconstituents using sulphuric acid. Upon hydrolysis, the ORC breaks downto glucuronic acid (approximately 80%) and glucose (20%). The glucuronicacid residues then undergo a colour reaction with carbozole, theabsorbance of which is measured against a series of ORC standards togive an estimation of the ORC content.

[0073] Samples of the material under test (10 mg) are placed inhydrolysate tubes. De-ionised water (0.5 ml) and concentrated sulphuricacid (3 ml) are added, and the mixture is mixed on a vortex mixer for 15minutes and checked for complete dissolution of the sample.

[0074] An aliquot (0.1 ml) of each sample hydrolysate is added to 2.9 mlof sodium tetraborate solution (0.025 molar in concentrated sulphuricacid) and mixed using vortex mixer. The sample tubes are placed in aboiling water bath for 10 minutes, and then cooled. Then 0.1 ml ofcarbozole solution (0.125% in ethanol) is added to each tube and mixedthoroughly with a vortex mixer, followed by placing the tubes in aboiling water bath for 15 minutes and cooling. The absorbance of theresulting solutions at 523 nanometers is then measured against a zeroconcentration ORC standard.

[0075] Procedure 4

[0076] The number of bacteria, fungi or yeast organisms present in thematerials according to the present invention is measured as follows.

[0077] A 2 gm sample of the material is extracted with 100 ml of sterileone-quarter strength Ringer's solution, and an aliquot (5 ml) is passedto a sterile membrane filter (pore size 0.45 μm) for sterile filtration.The filters are placed onto a nutrient medium in a Petri dish, andincubated under sterile conditions for 48 hours at 30° C. to allow thegrowth of germ colonies which can be counted with the naked eye or undera stereomicroscope if necessary. Appropriate control blanks are alsorun.

[0078] The level of microbiological contamination of the samples isexpressed as the total viable count (TVC) in colony forming units pergram (cfu/g) in accordance with the following formula:

TVC=[(N×100)/(5×W)]  

[0079] where N is the count of colonies, W is the weight of the samplein grams, 100 is the volume of the extractant solution in ml, and 5 isthe volume of the aliquot (5 ml) that is filtered.

[0080] Procedure 5

[0081] The wet and dry tensile strengths of the material according tothe invention are measured as follows.

[0082] Samples are die cut from a 3 mm thickness pad of the material.The sample dimensions are 2.5×12 cm. The samples are loaded into anInstron tensile tester with jaw face dimensions 50×25 ml. The drytensile strength is measured as the load at 20% elongation and the loadat break. The extension at break is expressed as the percentage of theinitial jaw separation. A minimum of 5 specimens was tested.

[0083] The wet tensile measurements are carried out in the same way onsamples that have been soaked for 15 minutes in phosphate bufferedsaline (PBS).

[0084] Procedure 6

[0085] The pH of solid materials according to the present invention ismeasured by macerating 100 gm of the material in 100 ml of deionisedwater, and measuring the pH of the resulting slurry with a glasselectrode.

[0086] Procedure 7

[0087] The resorption rate of the composite materials according to theinvention is measured in a flow of simulated wound fluid as follows.

[0088] A circular pad of the material under test, of thickness 3 mm anddiameter 6 cm is placed in a cylindrical recess and covered with a layerof liquid-impermeable backing material. Simulated wound fluid (3.45 mg/lcollagenase in phosphate buffered saline) was pumped radially at a rateof 2.5 ml, 7.5 ml or 12 ml/24 hours from an opening below the centre ofthe disk under test to six openings disposed radially around the edgesof the disk under test, below the disk under test, to simulate low,medium and high wound exudate flow rates. The time to resorption wasestimated as the time required for complete dissolution of the pad undertest. This time was at least two days for the high flow rate, at leastthree days for the medium flow rate, and at least six days for the lowflow rate.

[0089] Procedure 8

[0090] The liquid absorbency of the materials according to the presentinvention was measured as follows.

[0091] A sample (typically 2.5 cm×2.5 cm×0.3 cm) of the material undertest was weighed dry, and then immersed in phosphate buffered saline(PBS) for 15 minutes, removed with tweezers, and weighed again. Theliquid absorbency was then calculated in grams of absorbed liquid pergram (dry weight) of the material.

[0092] Procedure 9

[0093] The levels of bacterial endotoxin in the materials according tothe present invention are determined as follows.

[0094] Briefly, endotoxins from gram-negative bacterial cell walls causelimulus amebocyte lysate (LAL) to gel. The test is conducted as a limittest, wherein the sample is determined to be positive or negative to thetest, judged against pre-established endotoxin concentrations. Positiveand negative controls are essential and are carried out with each testrun. The control standard endotoxin has its potency verified againstreferenced standard endotoxin (USP EC6). Details of the method can befound in Carl Freudenberg Method 091,102 (pyrogenicity); in USP XXIII(1985); in the FDA Guidelines 1987 and in European Pharmacoepia 2.6.14(1998).

[0095] The embodiment of Example 1 has been described by way of exampleonly. Many other compositions and methods falling with the scope of thepresent invention will be apparent to the skilled reader.

1. A sterile freeze-dried sponge, wherein at least 80% by weight of thesponge consists of a mixture of collagen and oxidized regeneratedcellulose in the weight ratio 60:40 to 40:60, and wherein the sponge hasa dry tensile strength as herein defined of more than 3N.
 2. A sterilefreeze-dried sponge, wherein at least 80% by weight of the spongeconsists of a mixture of collagen and oxidized regenerated cellulose inthe weight ratio 60:40 to 40:60, wherein the sponge has a wet tensilestrength as herein defined greater than 1N.
 3. A sterile freeze-driedsponge according to claim 1 , wherein the sponge is substantially freeof chemical cross-links.
 4. A sterile freeze-dried sponge according toclaim 1 , wherein the collagen has a degree of denaturation less than20%.
 5. A sterile freeze-dried sponge according to claim 5 , wherein thecollagen has a degree of denaturation less than 10%.
 6. A sterilefreeze-dried sponge according to claim 4 , wherein 80% of the oxidizedregenerated cellulose fibers by volume are between 20 μm and 1000 μm inlength.
 7. A sterile freeze-dried sponge according to claim 6 , whereinthe mean length of the oxidized regenerated cellulose fibers is from 250to 450 μm.
 8. A sterile freeze-dried sponge according to claim 1 ,wherein the oxidized regenerated cellulose is in the form of fibers, andat least 90% of the fibers by volume are less than 1 mm in length.
 9. Asterile freeze-dried sponge according to claim 1 , wherein the ratio ofcollagen to oxidized regenerated cellulose is from 50:50 to 55:45.
 10. Asterile freeze-dried sponge according to claim 1 , wherein the bioburden(TVC) of the sponge is less than 100 cfu/g, preferably less than 10cfu/g, more preferably less than 1 cfu/g.
 11. A sterile freeze-driedsponge according to claim 1 , wherein the sponge contains from 5 to 15%by weight of water.
 12. A sterile freeze-dried sponge according to claim1 , wherein a layer of the sponge of thickness 3 mm has an uncompressedabsorption capacity of 0.9% saline of from 15 to 20 g/100 cm².
 13. Asterile freeze-dried sponge according to claim 1 , wherein the spongehas a resorption time under simulated physiological conditions of morethan 48 hours.
 14. A method of manufacture of a freeze-dried sponge padcomprising the steps of:— providing an acidified paste of purifiedcollagen fibers, wherein the collagen is less than 10% denatured;providing oxidized regenerated cellulose fibers, wherein at least 80% ofsaid fibers have lengths in the range of 20 μm to 1000 μm; combiningsaid collagen and said ORC fibers in a homogeneous aqueous dispersion ina weight ratio of 60:40 to 40:60 collagen:ORC, said aqueous dispersionbeing acidified to a pH in the range of 2.8 to 3.2 and having a totalsolids concentration of 0.8 to 1.2% by weight; pouring said aqueousdispersion into trays to a depth greater than 1 cm; freezing thedispersion to a temperature below −30° C., followed by a temperatureprogrammed freeze drying and dehydrothermal cross-linking to a finalmoisture content of 5-15% by weight; splitting the freeze-drieddispersion to remove surface layers and leave one or more pads; andsterilizing the one or more pads by gamma-irradiation.
 15. A methodaccording to claim 14 carried out substantially without the use of anychemical cross-linking agents.
 16. A method according to claim 14 ,wherein the step of providing collagen comprises the following steps:—providing fresh and unswollen splits of bovine corium; treating thesplits with a hypochloride solution to inhibit microbial activity;treating the corium with a solution containing sodium hydroxide andhydrogen peroxide to swell and sterilize the corium; then treating thecorium with a aqueous alkali solution at a pH greater than 12 andtemperature less than 50° C. for a period of ten-fourteen days; thentreating the corium with a aqueous acid solution at a pH of 0.8-1.2 andtemperature less than 50° C.; then washing the corium, and comminutingthe corium with sufficient water to form a paste.
 17. A method accordingto claim 14 , wherein the step of providing oxidized regeneratedcellulose fibers comprising milling an oxidized regenerated cellulosecloth and screening the milled particles to remove particles having sizeless than 20 μm or greater than 1000 μm.
 18. A method according to claim14 , wherein the step of dispersing the collagen and the ORC comprisesthe steps of:— adding an acid-swollen collagen/water paste to acidifiedwater; adding oxidized regenerated cellulose fibers to the acidifiedwater; and homogenizing the resulting mixture.
 19. A method according toclaim 14 , wherein the step of freezing is carried out by placing thetrays containing the aqueous dispersion onto chilled shelves in afreezer followed by holding the trays at a temperature below −30° C.until the freezing is complete.
 20. A method according to claim 14 ,wherein the step of freeze drying is carried out with dehydrothermalcross-linking.
 21. A method according to claim 14 , wherein the step ofsterilizing is carried out by gamma-irradiation at a dose of 18-29 KGy.22. A method according to claim 14 , wherein the weight ratio ofcollagen to oxidized regenerated cellulose is from 50:50 to 55:45 andthe pH of the aqueous dispersion is from 2.9 to 3.1.